Haloferax volcanii as immobilised whole cell biocatalyst: new applications for halophilic systems

Abstract

Enzyme-mediated synthesis of pharmaceutical compounds is a 'green' alternative to traditional synthetic chemistry, and microbial engineering opens up the possibility of using whole cells as mini-factories. Whole-cell biocatalysis reduces cost by eliminating expensive enzyme purification and cofactor addition steps, as well as resulting in increased enzyme stability. Haloferax volcanii is a model halophilic archaeon encoding highly salt and organic solvent tolerant enzymes such as alcohol dehydrogenase (HvADH2), which catalyses the reduction of aldehydes and ketone in the presence of NADPH/NADH cofactor. A H. volcanii strain for constitutive HvADH2 expression was generated using a strong synthetic promoter (p.syn). The strain was immobilised in calcium alginate beads and repeatedly used as a whole-cell biocatalyst. The reduction of acetophenone, used as test substrate, was very successful and high yields were detected from immobilised whole cells over repeated biotransformation cycles. The immobilised H. volcanii retained stability and high product yields after 1 month of storage at room temperature. This newly developed system offers halophilic enzyme expression in its native environment, high product yield, stability and reusability without the addition of any expensive NADPH/NADH cofactor. This is the first report of whole cell-mediated biocatalysis by the halophilic archaeon H. volcanii.

Keywords: Biocatalysis; Biocatalyst; Biotransformation; Haloferax volcanii; Whole cell immobilisation.

Fig. 1

Bioconversion of acetophenone to 1-phenylethanol catalysed by HvADH2. In the presence of NADPH/NADH cofactor, acetophenone is reduced to 1-phenylethol by the activity of HvADH2 enzyme

Fig. 2

Map of constitutive gene expression system in H. volcanii.a pTA1992. The p.syn promoter is used for constitutive expression of His (Histidine) tagged proteins in H. volcanii. A multiple cloning site is located after His tag, and is flanked by L11e and t.Syn terminators to prevent read-through transcription. pTA1992 was transformed into H1325 to generate H3925 to serve as empty vector control for biotransformation, b pTA2035. The adh2 (HVO_B0071, 1050 bp) gene was inserted in pTA1992 under the control of the constitutive p.syn promoter. pTA2035 was transformed into H1325 to generate H3924 for HvADH2 expression

Fig. 3

HvADH2 expression and enzymatic activity from H3924. a SDS-PAGE confirmation of high HvADH2 expression from H3924, lane 1, H3924 His-HvADH2 cell lysate (HvADH2 indicated in red box); lane 2, purified HvADH2 (37.8 kDa); lane 3, H3925 cell lysate, b purified HvADH2 protein concentration and enzyme specific activity, mean ± SD, n = 5

Fig. 4

Procedure for immobilising H. volcanii within calcium alginate beads. aH. volcanii cell pellet resuspended in YPC broth was mixed gently with 4% sodium alginate solution (in dH₂O) using a magnetic stirrer. Using a BD Plastipak^TM syringe, the mixture was added dropwise into 1.5% CaCl₂ solution to form beads, b and c formation of pink beads confirmed entrapment of H. volcanii within calcium alginate beads, the distinctive colour is due to presence of high carotenoid pigment

Fig. 5

Characterisation of conditions for maximal product yield. a Nutrient broth. Biotransformation reactions were performed in YPC, YPC + 4% fructose, YPC + 4% lactate, YPC + 4% sucrose, 2 × YPC⁺, YPC + 4% glucose, n = 3. b Temperature. Biotransformation was performed at 25 °C and 45 °C, n = 3. c Agitation speed. Beads in broth were agitated at 50 rpm, 100 rpm, 150 rpm and 200 rpm speed, n = 5. d Substrate concentration. 10 mM and 50 mM acetophenone substrate was added to the broth, n = 3. e Inducible vs constitutive gene expression system. Biotransformation was performed using the inducible (+ 5 mM tryptophan) adh2 strain H1332 and the constitutive adh2 strain H3924, n = 3, mean ± SD

Fig. 6

Repeated batch production of 1-phenylethanol by immobilised H. volcanii in calcium alginate beads. Product yield was measured by HPLC each day over a successive period of 12 days (cycles). After 24 h, the entire YPC + 4% glucose broth containing beads was decanted, beads were washed twice with 18% salt water and the biotransformation cycle was started again by adding fresh batch of broth supplemented with 5 mM acetophenone substrate. Data represents mean ± SD, n = 3

Fig. 7

Calcium alginate bead morphology is unperturbed after 12 successive biotransformation cycles. Six beads with immobilised H. volcanii were randomly selected from each biological replicate before (a) and after 12 biotransformation cycles (b). Diameter of the beads were measured, mean ± SD, n = 6